Production of recombinant Von Willebrand factor by CHO cells cultured in macroporous microcarriers

Recombinant Chinese hamster ovary cells producing Von Willebrand factor have been successfully grown in gelatin macroporous microcarriers (Cultispher-G). Serum-free cultures were maintained in 1, 4, and 10 liter fermentors for more than two months. Comparative studies with Cytodex-3 microcarriers have been performed in 1 liter fermentors. The lower specific Von Willebrand factor productivity of CHO cells cultivated on Cultispher-G were offset by higher cell densities (10⁷–2×10⁷ cells/ml). Volumetric Von Willebrand factor productivity was influenced by oxygen concentration, and remained stable during scale-up from 1 to 10 liter fermentors.     

A new recombinant procoagulant protein derived from the cDNA encoding human factor VIII

We have constructed new B domain deletion derivatives of human factor VIII (FVIII) by manipulating the cDNA using recombinant DNA techniques. One of these new derivatives, FVIII delta II, in which amino acids 771(pro)-1666(asp) have been deleted, no longer contains the protease cleavage site at amino acid position 1648(arg)-1649(glu) known to be involved in the initial step of FVIII processing. We have expressed this molecule in both baby hamster kidney (BHK) 21 cells using the vaccinia virus (VV) expression system and have established Chinese hamster ovary (CHO) derived permanent cell lines expressing either recombinant (r)FVIII or FVIII delta II. The characteristics of FVIII delta II have been compared to those of rFVIII and/or plasma derived (pd) FVIII. FVIII delta II has the following properties: (i) it exhibits FVIII procoagulant activity; (ii) it is expressed at 5-fold higher levels than is the complete molecule in comparable systems; (iii) it migrates for the most part as a single major band on SDS-PAGE, in contrast to the complete molecule; (iv) it is activated to a greater extent by thrombin than is either rFVIII or pdFVIII; and (v) it retains the ability to bind von Willebrand factor (vWf).     

CD3/Ti gamma A: a functional gamma-receptor complex expressed on human peripheral lymphocytes

We have recently developed a mAb designated anti-Ti gamma A, which was found to immunoprecipitate from the well characterized CD3+ TCR alpha/beta- F6C7 fetal clone a CD3-associated disulfide-linked gamma-glycoprotein. This antibody recognizes approximately 3% of adult peripheral lymphocytes and delineates a CD2+ CD3+ TCR alpha/beta- CD4- NKH1- subset where expression of CD8 appears to vary widely from one individual to another. In the present study, we have used anti-Ti gamma A mAb to assess whether gamma-chains expressed on these adult lymphocytes are used as functional R. The two activities which have been associated thus far with TCR gamma+ cells, that is, IL-2-dependent proliferation and non-MHC-restricted cytotoxicity, were investigated here by using either resting or activated Ti gamma A+ lymphocytes. On the resting state, these cells (which appear as a very homogeneous population of granular lymphocytes) mediate little if any NK activity that could not be augmented by anti-Ti gamma A mAb. In contrast, after initial stimulation by PHA plus rIL-2 and subsequent culture in the presence of IL-2, activated Ti gamma A+ lymphocytes were strongly lytic against a series of conventional NK target cell lines. This cytotoxic function was either blocked or enhanced by anti-Ti gamma A mAb, depending upon experimental conditions. With respect to proliferation, it was possible to induce responses of resting Ti gamma A+ lymphocytes with antibody-coated CNBr beads only in the presence of exogenous IL-2, whereas, in culture, the same cells proliferated directly and secreted IL-2 after treatment by anti-Ti gamma A beads. Taken together, these data demonstrate that a major subset of circulating CD3+ TCR alpha/beta- lymphocytes use protein products of T cell gamma rearranging genes as functional R structures.     

Late Holocene sea level indicators from twelve atolls in the central and eastern Tuamotus (Pacific Ocean)

New sea-level data for the late Holocene period are reported from twelve atolls of the Tuamotu archipelago: Faaite, Hikueru, Marokau, Hao, Amanu, Tatakoto, Pukarua, Nukutavake, Vairaatea, Tureia, Nukutipipi, and Hereheretue. The data come from coral conglomerate outcrops, coral colonies in growth position, in situ reef framework and marine notches; they give consistent results, and their ages are controlled by 29 radiocarbon datings. The Holocene MSL remained 0.8±0.2 m higher than at present, from before 4000 years BP until at least 1500 years BP, then dropped gradually to the present level. This pattern is very similar to that reported from the northwest Tuamotus, suggesting the absence of measurable differential vertical movements over a distance greater than 1300 km during the late Holocene. Effects of the 1983 cyclones on reef morphology are reported from some atolls and radiocarbon dates of some storm-generated reef blocks are given.     

Parenteral supplementation with zinc in surgical patients corrects postoperative serum-zinc drop

Zinc has been known for a long time to facilitate wound healing. But, so far, supplementation trials in patients treated by major severity surgery gave either partial or controversial results. In a double-blind, randomized study including 30 patients, we show that zinc supplements (30 mg/d for 3 d) administered by a drip correct postoperative drop of serum zinc, that this correction concerns the available part of serum zinc (i.e., zinc that is bound to compounds other than alpha-2 macroglobulin in serum), and that this supplementation can improve clinical wound healing. Possible influence of increased urinary output after the intervention is discussed, and we found that serum cortisol remains stable when zinc/albumin ratio is stable, and increases sharply when the same ratio drops. Cortisol, therefore, seems to play a major role in zinc redistribution after surgery.     

Characterisation of two differently processed forms of human recombinant factor IX synthesised in CHO cells transformed with a polycistronic vector

A stable transformed cell line constitutively expressing human factor IX has been established. Wild-type Chinese hamster ovary cells (CHO cells) were transformed using a polycistronic expression vector carrying a previously isolated factor IX cDNA and a selection gene encoding the Escherichia coli xanthine-guanine phosphoribosyl transferase. One clone, CHO 622.4, contains a high number of genomically integrated plasmids and secretes 1-3 mg factor IX l-1 day-1 into the culture medium with a biological activity ranging from 25% to 40%. The recombinant molecule was purified either by conventional chromatography or by immunoaffinity chromatography using antibodies specific to a calcium-induced factor IX conformer. The purified recombinant protein migrates as a single band with the same mobility as that of natural factor IX on SDS/polyacrylamide gels. N-terminal sequencing shows tow differently processed forms of recombinant factor IX: whereas the majority of the zymogen is correctly processed, approximately 20% of the purified recombinant molecule contains an 18-amino-acid NH2-extension corresponding to the precursor form of factor IX. Analysis of the 4-carboxyglutamic acid content indicates a high but incomplete carboxylation (70%) of the recombinant molecule as compared to natural factor IX. The carbohydrate composition of both the natural and recombinant molecules has been determined. Both molecules have a N-glycan structure of similar complexity, indicating that factor IX contains all the information to direct the same glycosylation pattern in human liver cells and in an unrelated cell line such as CHO-K1.     

Evolution of the primate beta-globin gene region. High rate of variation in CpG dinucleotides and in short repeated sequences between man and chimpanzee

A 5500 base-pair fragment including the beta-globin gene downstream from codon 122 and about 4000 base-pairs of its 5' flanking sequence was cloned from chimpanzee DNA and thoroughly sequenced before being compared with the corresponding human sequence: 88 point differences (83 substitutions and 5 deletions or insertions of 1 base-pair) were detected as well as seven more important deletion/insertion events. These changes occur preferentially in two kinds of structure. First, 40% of the CpG dinucleotides present in either human or chimpanzee sequences are affected by nucleotide variations. This corresponds to a divergence level considerably higher than that expected. Second, most short repeated sequences found in the 5' extragenic sequence are involved in mutational events (amplification or contraction of the number of basic motifs as well as point substitutions or deletions/insertions of 1 base-pair). Considering the very low level of nucleotide sequence divergence between these two closely related species, our data provide direct evidence for CpG and tandem array instability.